In stead of the conventionally used organic chemical reagents, reagents developed making use of enzyme reactions have been broadly applied in recent years to the inspection and diagnosis of morbid states.
These enzyme reaction-aided reagents use the property of enzymes to convert certain components in the living body into detectable substances in specific fashion, that is, they are used generally in a process in which a substance to be measured (A) is converted into an intermediate product (I-1) using an enzyme (a) specific for the substance, further converted into an intermediate product (I-2) by the action of an enzyme (i-1) specific for the intermediate product (I-1) and finally converted into a detectable substance (F) by repeating these steps, and then the detectable substance (F) is quantitatively measured based on the changes in color tone by using a spectrophotometer or by comparing and matching colors with references. ##STR1##
In these reagents, NAD or its reduced form (NADH) and its analogous compound NADP or its reduced form (NADPH) are broadly used as the detectable substance (F) (when these compounds are named generically, they are referred to as "nicotine nucleotides" hereinafter), and these nicotine nucleotides show changes in their absorbance at an ultraviolet region (around 340 nm), so that a reagent in which formed or decreased nicotine nucleotides are measured using a spectroscope has been proposed. Also, a number of reagents have been reported in which a tetrazolium compound is converted into its corresponding formazan by the action of a diaphorase upon formed NADH or NADPH and the resulting formazan is measured at a visible region (e.g., bile acid (JP-A-60-214900; the term "JP-A" as used herein means an "unexamined published Japanese patent application") and Clinical Chemistry (a Japanese journal), vol. 19, pp. 290-299 (1990)), triglyceride (JP-A-55-14899), alcohol (JP-B-4-3947; the term "JP-B" as used herein means an "examined Japanese patent publication"), amylase (JP-B-63-37640), creatine kinase (JP-A-58-16699), NAD(P)H (JP-B-4-70000), polyamine (JP-B-6-68490), glucose (JP-B-7-34757) and benzylamine (JP-A-7-184693)). In addition, test strips for the measurement of creatine kinase activity have also been reported (JP-A-63-283600, JP-B-6-95959 and JP-A-1-320999).
However, these reagents use dichlorophenolindophenol (to be referred to as "DCIP" hereinafter), Tetrazolium Blue, Neotetrazolium Blue, MTT, INT or Nitrotetrazolium Blue as the color reagent.
Among these color reagents, DCIP is not suitable for the preparation of test strips, because its colored state by the action of a diaphorase changes into colorless state.
Also, the tetrazolium compounds used as the other color reagents have low solubility in water so that test strips cannot be prepared easily (cf. Comparative Examples below) and, even if it is processed into the form of a test strip, sufficient measuring range cannot be obtained so that additional steps such as dilution of the samples to be measured and the like are required (e.g., Journal of medical Technology, vol. 11. No. 6, pp. 496-505, describes that creatine kinase activity can be measured only up to 1,000 units/l even when samples are diluted by a factor of 9).
In addition, since the samples to be tested such as blood, urine and the like are aqueous substances, such test strips have a problem in that measurement of the activity with high sensitivity and good reproducibility is extremely difficult to effect even if reflected light of a sample to be tested is measured by directly adding it to the test strip.
On the other hand, with regard to other cases than creatine kinase and the like enzymes, there is a case in which a reducing type color former is used in test strips for the measurement of magnesium as an inorganic substance, and tetrazolium salts are cited as examples of the reducing type color former (e.g., JP-A-9-266796 and JP-A-9-266797), but only Tetrazolium Violet among the exemplified tetrazolium salts is a tetrazolium salt having high water-solubility and all of the rest have low solubility in water. In addition, since spontaneous coloring occurs when other typical water-soluble tetrazolium compounds are used in such test strips, it has been considered that the use of water-soluble tetrazolium compounds in such test strips as color reagents is not appropriate.